Genomics, 1992 May, 13(1), 75 - 80 Sequence-tagged sites (STSs) spanning 4p16.3 and the Huntington disease candidate region; Gusella JF et al.; The generation of sequence-tagged sites (STSs) has been proposed as a unifying approach to correlating the disparate results generated by genetic and various physical techniques being used to map the human genome . We have developed an STS map to complement the existing physical and genetic maps of 4p16.3, the region containing the Huntington disease gene . A total of 18 STSs span over 4 Mb of 4p16.3, with an average spacing of about 250 kb . Eleven of the STSs are located within the primary candidate HD region of 2.5 Mb between D4S126 and D4S168 . The availability of STSs makes the corresponding loci accessibility to the general community without the need for distribution of cloned DNA . These STSs should also provide the means to isolate yeast artificial chromosome clones spanning the HD candidate region. Mol Cell Biol, 1992 May, 12(5), 2295 - 301 A dominant negative allele of p34cdc2 shows altered phosphoamino acid content and sequesters p56cdc13 cyclin; Fleig UN et al.; The cdc2 gene product, a 34-kDa phosphoprotein with serine/threonine protein kinase activity, has been implicated as the key component in the regulation of the eucaryotic cell cycle . Activation of the cdc2 protein kinase is regulated by its phosphorylation state and by interaction with other proteins . We have mutagenized the fission yeast cdc2 gene to obtain conditionally dominant negative alleles . One of these mutants, named DL2, is characterized in this report . Overexpression of the mutant protein in a wild-type cdc2 background is lethal and leads to arrest in the G2 phase of the cell cycle . The mutant phenotype is the result of a single amino acid change in the GDSEID motif of the protein, a region of identity in all cdc2 homologs, and results in a nonfunctional protein that shows an altered content of phosphothreonine . Multicopy suppressors of the dominant negative phenotype have been isolated, and one of these has been shown to encode the cdc13 cyclin B gene product. Biol Mass Spectrom, 1992 May, 21(5), 242 - 8 Biosynthesis and characterization of 3-hydroxyalkan-2-ones and 2,3-alkanediols: potential products of aldehyde metabolism; Montgomery JA et al.; A mass spectrometric study of an enzymatic synthesis of 3-hydroxyalkan-2-ones (acyloins) is presented . Incubation of pyruvate or (13C3)pyruvate and various alkanals in the presence of pig heart pyruvate dehydrogenase or yeast pyruvate decarboxylase resulted in the formation of acyloins with chains two carbons longer than the alkanals . Product formation was rapid for all saturated aldehydes with chain lengths from 2 to 12 carbons . Incubation with 2,3-unsaturated aldehydes did not produce condensation products . Reduction of acyloins with sodium borohydride produced the corresponding 2,3-alkanediols . Analysis by gas chromatography/mass spectrometry was used to characterize the 3-hydroxyalkan-2-ones as the oxime-trimethylsilyl derivatives and the 2,3-alkanediols as the bistrimethylsilyl derivatives. New Biol, 1992 May, 4(5), 512 - 9 Analysis of the DNA binding and transcriptional activation properties of the Ets1 oncoprotein; Gegonne A et al.; The c-ets1 gene product (Ets1) is the prototype of a family of sequence-specific transcriptional activators which have been implicated in various developmental processes and in the response of cells to a variety of extracellular stimuli . We report here a structure-function analysis of the DNA binding and transcriptional activation properties of Ets1 . The minimal region required for specific DNA binding is located at the carboxy-terminus of Ets1, a domain highly conserved in all known members of the Ets family . Transcriptional activation by Ets1 in mammalian cells requires an additional domain of 110 amino acids characterized by a high content of acidic residues and localized in the amino-terminal half of the protein . This domain also functions as a transcriptional activation domain in yeast cells when linked to the heterologous DNA binding domain of Gal4 . In contrast to its conservation in Ets1 proteins across vertebrate species, this activation domain is not conserved in other members of the Ets family . These results indicate that an important level of specificity between different members of the Ets family may reside in the differential interactions of their respective activation domains with distinct general transcription factors or different associated coactivators. Ann Hum Genet, 1992 May, 56 ( Pt 2), 93 - 7 Carrier detection of deletions of the Hunter gene by in situ hybridization; Stone S et al.; Deficiency of the lysosomal enzyme alpha-iduronate sulphate sulphatase (IDS) causes the clinical manifestations of Hunter syndrome, an X-linked condition . In about 30% of male patients, the disease is due to a major deletion . Using a non-isotopic in situ hybridization (NISH) method, and a yeast artificial chromosome (YAC) probe, the Hunter gene was mapped to the terminal region of the human X chromosome, close to the Xq28 band . The NISH procedure was then applied to investigate the carrier status of female relatives of a Hunter patient known to have a deletion of the IDS gene . Unequivocal evidence that two female relatives were carriers of the deletion was obtained, demonstrating that the NISH method is a valuable diagnostic tool in genetic counselling of families with Hunter patients. J Natl Med Assoc, 1992 May, 84(5), 449 - 52 Localized pulmonary disease due to Trichosporon beigelii; Qadri SM et al.; A case of pulmonary infection caused by Trichosporon beigelii is reported . The infection occurred in a neutropenic patient with acute lymphoblastic leukemia . His chest radiograph showed a 6-cm pulmonary infiltrate in the right midzone and an apical infiltrate on the left . Repeated cultures of bronchoalveolar lavage grew budding yeast that was identified as T beigelii on the basis of morphological, cultural, and biochemical characteristics . He responded to amphotericin-B therapy . Systemic infections caused by this yeast are rare and its causal relationship in localized lung disease has been reported only seven times previously. Srp Arh Celok Lek, 1992 May-Jun, 120(5-6), 184 - 7 {Pityriasis versicolor--modern views on etiology, pathogenesis and therapy}; Karadaglic Dj; Pityriasis versicolor (Tinea versicolor) is a superficial chronic fungal infection caused by Pityrisporum species which are normal "inhabitants" of the cutaneous flora . The morphologic changes from yeast to mycelial hypha form are important in the development of clinical lesions . The onset and course of the disease are under the influence of genetic factors, age, sex, climate, local environmental factors, malnutrition, pregnancy, oral contraceptives, corticosteroid and immunosuppressive treatment . They favorize transformation of saphrophytic to pathogenic form, and are the cause of recurrences and chronicity of the disease . The ultrastructural and immunologic studies which are carried out today would significantly contribute to a better understanding of pathogenesis of the disease . The disease is limited to seborrheic areas of the skin and commonly has three clinical forms: papulosquamous, follicular and inverse . This can make great problems in differential diagnosis . In the treatment of these patients it should be kept in mind that Pityriasis versicolor is not a primary contagious disease and that conversion of Pityrisporum depends on the predisposing factors . Numerous medicaments with local and systemic effect which are used nowaday in the treatment and prevention of pityriasis are reported . Way of application, doses, duration of therapy, advantages and disadvantages are given in detail. New Biol, 1992 May, 4(5), 551 - 7 FLP-mediated intermolecular recombination in the cytoplasm of Drosophila embryos; Konsolaki M et al.; We show that when a heat-shock-driven gene that encodes the yeast FLP recombinase is injected into preblastoderm Drosophila embryos, it promotes intermolecular recombination between two coinjected plasmids that bear the specific recombination target sequence, FRT . Minimal, 34-bp FRT sites in the two plasmids are sufficient for their cointegration . The reaction is efficient enough to produce detectable recombinants when one of the plasmids is present in as little as 1000 molecules per embryo . This is comparable to the concentration of unique chromosomal sites, raising the possibility that integration of injected plasmid DNA into FRT-bearing fly chromosomes may also be achievable . Since integrants might be stabilized against the reverse excision reaction if the recombinase could be provided in a sharp pulse, it is encouraging that efficient plasmid cointegration is also achieved when in vitro synthesized FLP RNA rather than DNA is injected into the embryos. EMBO J, 1992 May, 11(5), 1949 - 55 Transcription mapping of a 100 kb locus of Plasmodium falciparum identifies an intergenic region in which transcription terminates and reinitiates; Lanzer M et al.; We have mapped Plasmodium falciparum erythrocytic stage transcription units on chromosome 10 in the vicinity of the gene encoding the glycophorin binding protein (GBP130) using yeast artificial chromosomes (YACs) . Three erythrocytic stage transcription units are clustered in a 40 kb region . Two of these genes are closely linked, separated by less than 2 kb . Nuclear run-on data demonstrate that transcription of these two genes, though unidirectional, is monocistronic . Within this intergenic region are the sites at which transcription of the upstream gene terminates and the GBP130 gene initiates . These studies represent the first description of the minimal and necessary cis-acting elements for transcription termination and initiation in this protozoan parasite. Trends Genet, 1992 May, 8(5), 174 - 80 Ustilago maydis, the delightful blight; Banuett F; Recent studies of the corn smut fungus life cycle and its regulation by two mating type loci and other genes provide a cornucopia of challenges in cell biology, genetics and protein structure . The fungus can exist in two states: nonpathogenic and pathogenic . The change from one state to the other is accompanied by a change in morphology (yeast-like to filamentous) and growth properties (saprophytic to parasitic). Genomics, 1992 May, 13(1), 16 - 20 A 14-Mb physical map of the region at chromosome 11q13 harboring the MEN1 locus and the tumor amplicon region; Tanigami A et al.; We have constructed a physical map of chromosome 11q13, using 54 DNA markers that had been localized to 11q13.1----q13.5 by means of somatic hybrid cell panels . Although the map has some gaps, it spans nearly 14 Mb and includes the region containing the gene responsible for multiple endocrine neoplasia type 1 (MEN1) and also the region that is amplified in several types of malignant tumors . As the estimated average distance between each locus is roughly 300 kb, the markers reported here will be valuable resources for construction of contig maps with yeast artificial chromosomes and/or cosmid clones . Furthermore, these clones will be useful in efforts to identify the MEN1 gene and in analyses of the amplification units present at 11q13 in certain tumors. Biochem Biophys Res Commun, 1992 Apr 30, 184(2), 986 - 92 Contribution of the p51 subunit of HIV-1 reverse transcriptase to enzyme processivity; Huang SC et al.; Human immunodeficiency virus Type I reverse transcriptase is active as either the homodimer (p66/p66) or the heterodimer (p66/p51) . Purified recombinant p66 and p51 expressed in yeast were reconstituted in the presence of 60 mM sodium pyrophosphate to enhance dimer formation . Comparison of the processivity of these two active reconstituted forms shows that the heterodimer is more processive than the homodimer with a cycle almost twice as long as judged by assays utilizing poly (U,G) as a challenger to primer-template . Binding assays demonstrated that the heterodimer has a higher affinity for primer-template than the homodimer and that the p51 subunit has an affinity equal to that of the heterodimer . These results suggest that the p51 subunit functions to increase processivity in the heterodimer. Carbohydr Res, 1992 Apr 27, 228(2), 377 - 98 Chemical modification of the sugar part of methyl acarviosin: synthesis and inhibitory activities of nine analogues; Shibata Y et al.; Nine analogues of methyl acarviosin (1), the core structure of acarbose and its homologues, the 6-hydroxy-(2), 6-azido-(3), 6-amino- (4), 6-acetamido-(5), 6-methoxy-(6), 6-hydroxy-2-O-methyl-(8), and 6-hydroxy-3-O-methyl derivatives (9), including the 5-methoxycarbonyl analogue (7) and 3,6-anhydro derivative (10) of 2, were synthesized by chemical modification of the sugar part of 2 derived by condensation of methyl 3,4-anhydro-alpha-D-galactopyranoside (17) and 4,7:5,6-di-O-isopropylidenevalienamine (26) or by direct coupling between 26 and the 6-substituted methyl 3,4-anhydro-alpha-D-galactopyranoside derivatives . Compounds 2 and 8 show notable inhibitory activity against yeast alpha-D-glucosidase almost comparable to that of 1 . Introduction of a polar substituent at C-6 of 1 decreases the inhibitory activity . Interestingly, inversion of the conformation of the sugar part of 1 by introduction of the 3,6-anhydro bridge elicits almost no effect on the inhibitory activity. Nucleic Acids Res, 1992 Apr 25, 20(8), 1903 - 8 Kinetic trapping of H-DNA by oligonucleotide binding; Belotserkovskii BP et al.; Homopurine-homopyrimidine mirror repeats are known to adopt the H form under acidic pH and/or negative supercoiling . In H-DNA, one half of the purine strand enters the triplex whereas the second half is unstructured and can form duplex with complementary oligonucleotide . However, because the same oligonucleotide can form triplex with the homopurine-homopyrimidine insert, one could expect that oligonucleotide would make H-DNA thermodynamically less favorable, as was claimed by Lyamichev et al . Nucl . Acids Res . 16, 2165-2178 (1988) . Now we show that complex between oligonucleotide and H-DNA, formed under conditions favorable for the H-form extrusion, is kinetically trapped in superhelical DNA and remains stable up much higher pH values than H-DNA alone . Experiments on chemical probing show that such complex exists for a plasmid with native superhelical density at pH7 . We have also used this approach to demonstrate a pH-dependent structural transition in yeast telomeric sequence, d(CACACCCA)16. Nucleic Acids Res, 1992 Apr 25, 20(8), 1865 - 70 Efficient mischarging of a viral tRNA-like structure and aminoacylation of a minihelix containing a pseudoknot: histidinylation of turnip yellow mosaic virus RNA; Rudinger J et al.; Mischarging of the valine specific tRNA-like structure of turnip yellow mosaic virus (TYMV) RNA has been tested in the presence of purified arginyl-, aspartyl-, histidinyl-, and phenylalanyl-tRNA synthetases from bakers' yeast . Important mischarging of a 264 nucleotide-long transcript was found with histidinyl-tRNA synthetase which can acylate this fragment up to a level of 25% with a loss of specificity (expressed as Vmax/KM ratios) of only 100 fold as compared to a yeast tRNA(His) transcript . Experiments on transcripts of various lengths indicate that the minimal valylatable fragment (n = 88) is the most efficient substrate for histidinyl-tRNA synthetase, with kinetic characteristics similar to those found for the control tRNA(His) transcript . Mutations in the anticodon or adjacent to the 3' CCA that severely affect the valylation capacity of the 264 nucleotide long TYMV fragment are without negative effect on its mischarging, and for some cases even improve its efficiency . A short fragment (n = 42) of the viral RNA containing the pseudoknot and corresponding to the amino acid accepting branch of the molecule is an efficient histidine acceptor. J Biol Chem, 1992 Apr 25, 267(12), 8464 - 7 Mechanism of assembly of the RNA polymerase II preinitiation complex . Evidence for a functional interaction between the carboxyl-terminal domain of the largest subunit of RNA polymerase II and a high molecular mass form of the TATA factor; Conaway RC et al.; Genetic evidence argues that the highly conserved carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II functions directly in the regulation of transcription of many eukaryotic genes . The observation that partial deletion of the CTD of yeast RNA polymerase II reduces the ability of the enzyme to respond to signals from a variety of upstream activating sequences led to the proposal that the CTD plays a role in the dialogue between regulatory factors that bind upstream activating sequences and the "general" or "basal" transcription factors associated with RNA polymerase II at the promoter (Scafe, C., Chao, D., Lopes, J., Hirsch, J . P., Henry, S., and Young, R . A . (1990) Nature 347, 491-494) . Biochemical evidence for an interaction of the CTD with specific components of the basal transcription apparatus, however, has been lacking . To identify target(s) for CTD action, we probed steps in assembly of the RNA polymerase II preinitiation complex with monoclonal antibodies specific for the CTD . Our findings reveal a novel interaction of the CTD with a high molecular mass form of the TATA factor . This interaction occurs during binding of RNA polymerase II to its promoter and requires the action of additional basal transcription factors; it is not observed when the single-subunit yeast transcription factor IID serves as the TATA factor. J Biol Chem, 1992 Apr 25, 267(12), 8286 - 92 Biochemical analysis of the B subunit of the heteromeric CCAAT-binding factor . A DNA-binding domain and a subunit interaction domain are specified by two separate segments; Maity SN et al.; CCAAT-binding factors A (CBF-A) and B (CBF-B) are two subunits of the heteromeric CCAAT-binding factor . Portions of CBF-A and CBF-B have a high degree of amino acid sequence identity to segments of the HAP3 and HAP2 subunits of a yeast multimeric transcription factor . We show here that the subunits of CBF interact with each other in the absence of DNA binding . This interaction was revealed by cross-linking and coimmunoprecipitation studies . Both the DNA binding and subunit interaction functions of CBF-B have been examined by mutational analysis . A segment of 83 amino acids from residues 252 to 334, which corresponds to the evolutionarily conserved portion of CBF-B, is necessary and sufficient for CBF-A-dependent DNA binding . Carboxyl-terminal deletions of this segment (or mutations in arginine residues in this carboxyl-terminal part) abolish DNA binding, but do not alter subunit interactions between CBF-A and CBF-B . Mutations in hydrophobic amino acids within the amino-terminal part of the evolutionarily conserved sequence at positions 252-334 result in loss of both DNA binding and subunit interaction activities . Our results indicate that the evolutionarily conserved segment of CBF-B contains both DNA-binding and subunit interaction domains and that the integrity of both domains is essential for DNA binding. J Biol Chem, 1992 Apr 25, 267(12), 8270 - 4 Sequence requirements for precursor cleavage within the constitutive secretory pathway; Watanabe T et al.; We have recently demonstrated that the Arg-X-Lys/Arg-Arg sequence is a signal for precursor cleavage catalyzed by furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, within the constitutive secretory pathway . In this study, we further examined sequence requirements for the constitutive precursor cleavage by expression of various prorenin mutants with amino acid substitutions around the native Lys-Arg cleavage site in Chinese hamster ovary cells . The results delineate the following sequence rules that govern the constitutive precursor cleavage . (a) A basic residue (Lys or Arg) at the 4th (position -4) or 6th (position -6) residue upstream of the cleavage site besides basic residues at positions -1 and -2 is necessary . (b) At position -2, a Lys residue is more preferable than Arg . (c) At position -4, an Arg residue is more preferable than Lys . (d) At position 1, a hydrophobic aliphatic amino acid is not suitable. J Biol Chem, 1992 Apr 25, 267(12), 7979 - 82 Tyrosine codon corresponds to topa quinone at the active site of copper amine oxidases; Mu D et al.; The recently discovered organic cofactor of bovine serum amine oxidase, topa quinone, is an uncommon amino acid residue in the polypeptide backbone (Janes, S . M., Mu, D., Wemmer, D., Smith, A . J., Kaur, S., Maltby, D., Burlingame, A . L., and Klinman, J . P . (1990) Science 248, 981-987) . The amine oxidase gene from the yeast Hansenula polymorpha has been cloned and sequenced (Bruinenberg, P . G., Evers, M., Waterham, H . R., Kuipers, J., Arnberg, A . C., and Geert, A . B . (1989) Biochim . Biophys . Acta 1008, 157-167) . In order to understand the incorporation of topa quinone in eukaryotes, we have isolated yeast amine oxidase from H . polymorpha . Following protocols established with bovine serum amine oxidase, yeast amine oxidase was derivatized with {14C}phenylhydrazine, followed by thermolytic digestion and isolation of a dominant radiolabeled peptide by high pressure liquid chromatography . Comparison of resonance Raman spectra for this peptide to spectra of a model compound demonstrates that topa quinone is the cofactor . By alignment of a DNA-derived yeast amine oxidase sequence with the topa quinone-containing peptide sequence, it is found that the tyrosine codon, UAC, corresponds to topa quinone in the mature protein . In a similar manner, alignment of a tryptic peptide from bovine serum amine oxidase implicates tyrosine as the precursor to topa quinone in mammals. J Biol Chem, 1992 Apr 25, 267(12), 8679 - 84 Isolation and cloning of a voltage-dependent anion channel-like Mr 36,000 polypeptide from mammalian brain; Bureau MH et al.; A polypeptide of M(r) 36,000 (36 kDa) was isolated from detergent-solubilized membrane fractions of mammalian brain on a benzodiazepine affinity column utilized for the purification of the gamma-aminobutyric acid/benzodiazepine receptor protein, followed by preparative gel electrophoresis . Partial protein sequence for two fragments of the 36-kDa polypeptide allowed the isolation of cDNA clones from a rat hippocampal library . An open reading frame coding a sequence of 295 amino acid residues containing the two probe peptide sequences with minor differences, and a putative N-terminal signal peptide of 25 residues was found . Hydropathy index revealed no regions of alpha-helix suitable for membrane spanning, but several areas of alternating hydrophilic and hydrophobic residues consistent with beta-strands . The sequence of this brain protein was 24% identical to that of a yeast mitochondrial protein, the voltage-dependent anion channel (VDAC), and over 70% identical with the VDAC from human B lymphocytes . The gamma-aminobutyric acid type A (GABAA) receptor/36-kDa preparation purified on benzodiazepine affinity column has channel-forming activity in lipid bilayer membranes that is virtually identical to VDAC isolated from mitochondria of various sources, indicating that the 36-kDa protein is a new member of the VDAC family of proteins . An antiserum raised against the purified 36-kDa polypeptide was able to precipitate {3H}muscimol binding activity, indicating a tight association with the GABAA receptor protein in vitro and copurification on the benzodiazepine affinity column due to this association . Further studies are needed to determine whether such an association occurs in vivo. J Biol Chem, 1992 Apr 15, 267(11), 7904 - 10 Amino-terminal octapeptides function as recognition signals for the mitochondrial intermediate peptidase; Isaya G et al.; We have shown previously that cleavage of a number of precursors by the mitochondrial processing peptidase (MPP) requires an intermediate octapeptide (FXXSXXXX) between the MPP cleavage site and the mature protein amino terminus . We show now that these octapeptides, present at the amino termini of the intermediates, direct recognition of these substrates by the mitochondrial intermediate peptidase (MIP), leading to formation of mature proteins . Synthetic peptides, corresponding to the intermediate octapeptides of human ornithine transcarbamylase (OTC) and of Neurospora cytochrome c reductase Fe/S subunit (Fe/S), inhibit the processing activity of purified rat liver MIP in vitro, without affecting MPP activity; this indicates that the octapeptides can be recognized by MIP independent of the presence of the corresponding mature proteins and interact with a site that is crucial for MIP activity . MIP activity is not inhibited by a peptide lacking the amino-terminal hydrophobic residue, while substitution of such a residue by a polar amino acid causes a 10-fold reduction in the efficiency of MIP inhibition . To analyze the requirements for removal of the octapeptide from the intermediate proteins by MIP, artificial intermediates were synthesized and subjected to in vitro processing by purified MIP . The octapeptide can be cleaved by MIP only when the amino-terminal hydrophobic residue is also the amino terminus of the intermediate . Further, when the OTC octapeptide is joined to the mature amino terminus of another twice-cleaved precursor (pFe/S; rat malate dehydrogenase, pMDH), the chimeric intermediate is cleaved by MIP to the corresponding mature-sized protein . When the OTC octapeptide is joined to the mature amino terminus of a once-cleaved precursor (yeast F1-beta-ATPase, pF1-beta), however, this intermediate is not cleaved by MIP; rather, it is processed by MPP to mature-sized F1-beta . Therefore, amino-terminal octapeptides can be cleaved by MIP only within the structural context of twice-cleaved precursors. Eur J Biochem, 1992 Apr 15, 205(2), 537 - 43 M-phase-specific histone H1 kinase in fish oocytes . Purification, components and biochemical properties; Yamashita M et al.; We demonstrate, for the first time in fish, that a Ca(2+)-independent and cyclic-nucleotide-independent histone H1 kinase activity oscillates according to the cell cycle of the oocyte, peaking at the first and the second meiotic metaphase with a transient drop between them . The kinase, M-phase-specific histone H1 kinase (M-H1K), was purified from mature carp oocytes by using two exogenous substrates for assaying its activity: histone H1 and a synthetic peptide (SP peptide, KKAAKSPKKAKK) containing the sequence KSPKK, which includes the consensus sequence of the site phosphorylated by a serine/threonine-specific protein kinase encoded by the fission yeast cdc2+ gene (cdc 2 kinase) . The M-H1K and maturation-promoting factor (MPF) activities coincided closely throughout four steps of purification, strongly suggesting the identity of M-H1K and MPF . The final preparation was purified 5000-fold with a recovery of 4%, when histone H1 was used for the kinase assay, and 10,000-fold with a recovery of 7% when SP peptide was used . The purified molecular mass of the kinase was estimated to be 100 kDa by gel filtration and contained four proteins of 33, 34, 46 and 48 kDa . Anti-PSTAIR antibody recognizing cdc2 kinase cross-reacted with the 33-kDa and 34-kDa proteins, while the 46-kDa and 48-kDa bands cross-reacted with monoclonal antibodies raised against cyclin B . The 33-kDa protein was also recognized by an antibody against a goldfish cdk2 (Eg1) kinase, a cdc2-related kinase which has the PSTAIR sequence and binds to p13suc1 but does not form a complex with cyclin B . M-H1K activity corresponded well to the 34-kDa, 46-kDa and 48-kDa proteins but not to the 33-kDa protein . These results strongly suggest that M-H1K consists of cdc2 kinase forming a complex with cyclin B, and that cdk2 kinase is not a component of M-H1K, although it is found in the highly purified M-H1K . The purified M-H1K utilized Mg2+, Mn2+, ATP and GTP, and had a wide pH optimum ranging over 8.0-10.5 . The kinase was thermolabile and sensitive to freezing/thawing. FEBS Lett, 1992 Apr 13, 301(1), 23 - 8 Biochemical characterization of the p51 sub-unit of human immunodeficiency virus reverse transcriptase in homo- and heterodimeric recombinant forms of the enzyme; el Dirani-Diab R et al.; The biochemical properties of the p51 subunit of HIV-1 reverse transcriptase (RT) were studied in order to understand its role in the heterodimeric form p66/p51 found in virions . A recombinant form of RT, p51/p51, expressed in yeast, was purified and characterized . The enzyme was affinity labeled using a 5' modified oligonucleotide primer, covalently linked, that was further elongated in the presence of a radioactive dNTP precursor . We found that the p51 subunit was labeled in the p51/p51 form, thus reflecting its activity, while this subunit was catalytically silent in the heterodimer, since only the p66 subunit was labeled in the latter recombinant form . Processivity studies showed long-sized products synthesized by p51/p51, as in the case of the other RT forms . The effect of primer tRNA(Lys) on the p51/p51 activity showed a strong inhibitory effect in the absence of KCl, similar to that observed with the p66/p51 form, while the same p51/p51 enzyme was strongly stimulated by tRNA(Lys), like RT p66/p66, when KCl was present in the incubation mixture. Nucleic Acids Res, 1992 Apr 11, 20(7), 1457 - 62 DNA replication initiates at multiple sites on plasmid DNA in Xenopus egg extracts; Mahbubani HM et al.; Cell-free extracts of Xenopus eggs will replicate plasmid DNA molecules under normal cell cycle control . We have used the neutral/neutral 2-D gel technique to map the sites at which DNA replication initiates in this system . Three different plasmids were studied: one containing the Xenopus rDNA repeat, one containing single copy Xenopus genomic DNA, and another containing the yeast 2 microns replication origin . 2-D gel profiles show that many potential sites of initiation are present on each plasmid, and are randomly situated at the level of resolution of this technique (500-1000 bp) . Despite the abundance of sites capable of supporting the initiation of replication, pulse-chase experiments suggest that only a single randomly situated initiation event occurs on each DNA molecule . Once initiation has taken place, conventional replication forks appear to move away from this site at a rate of about 10nt/second, similar to the rate observed in vivo. Biochemistry, 1992 Apr 7, 31(13), 3472 - 7 Photoinduced electron transfer between cytochrome c peroxidase and horse cytochrome c labeled at specific lysines with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) Hahm S, Durham B, Millett F. The reactions of yeast cytochrome c peroxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)(bisbipyridine)ruthenium(II) {Ru(II)} were studied by flash photolysis . All of the derivatives formed complexes with cytochrome c peroxidase compound I (CMPI) at low ionic strength (2 mM sodium phosphate, pH 7) . Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in electron transfer to the ferric heme group in cytochrome c, followed by electron transfer to the radical site in CMPI . This reaction was biphasic and the rate constants were independent of CMPI concentration, indicating that both phases represented intracomplex electron transfer from the cytochrome c heme to the radical site in CMPI . The rate constants of the fast phase were 5200, 19,000, 55,000, and 14,300 s-1 for the derivatives modified at lysines 13, 25, 27, and 72, respectively . The rate constants of the slow phase were 260, 520, 200, and 350 s-1 for the same derivatives . These results suggest that there are two binding orientations for cytochrome c on CMPI . The binding orientation responsible for the fast phase involves a geometry that supports rapid electron transfer, while that for the slow phase allows only slow electron transfer . Increasing the ionic strength up to 40 mM increased the rate constant of the slow phase and decreased that of the fast phase . A single intracomplex electron transfer phase with a rate constant of 2800 s-1 was observed for the lysine 72 derivative at this ionic strength . When a series of light flashes was used to titrate CMPI to CMPII, the reaction between the cytochrome c derivative and the Fe(IV) site in CMPII was observed . The rate constants for this reaction were 110, 250, 350, and 140 s-1 for the above derivatives measured in low ionic strength buffer. Cell, 1992 Apr 3, 69(1), 173 - 84 chickadee encodes a profilin required for intercellular cytoplasm transport during Drosophila oogenesis; Cooley L et al.; The entire cytoplasmic contents of 15 highly polyploid nurse cells are transported rapidly to the oocyte near the end of Drosophila oogenesis . chickadee is one of a small group of genes whose mutant phenotype includes a disruption of this nurse cell cytoplasm transport . We have cloned the chickadee gene and found that cDNA clones encode a protein 40% identical to yeast and Acanthamoeba profilin . The nurse cells from chickadee egg chambers that lack ovary-specific profilin fail to synthesize cytoplasmic actin networks correctly . In addition, the nurse cell nuclei in chickadee egg chambers become displaced and often partially stretched through the channels leading into the oocyte, blocking the flow of cytoplasm . We suggest that the newly synthesized cytoplasmic actin networks are responsible for maintaining nuclear position in the nurse cells. Int J Immunopharmacol, 1992 Apr, 14(3), 391 - 7 Pre-clinical toxicity of IL-4: a model for studying protein therapeutics; Dean JH et al.; The purpose of this presentation was to review issues and findings in the pre-clinical development and evaluation of recombinant human protein therapeutics . Since human cytokines and lymphokines are endogenous proteins, their pre-clinical development and evaluation would seem straightforward and their toxicities minimal . Unfortunately, the pre-clinical development of this class of agents has been problematic and confounding . Some of the clinical toxicities and pharmacodynamics have been predicted by the pre-clinical evaluation and others have not . Some molecules are species specific which limits species selection for pre-clinical evaluation . Other confounding issues include: route of exposure, synergy of toxicity with other lymphokines, length of study design, immunogenicity, predictiveness of pre-clinical evaluation and iatrogenic toxicities . An approach used by SWPRD in the evaluation of this class of molecules was discussed . Insight gained during the pre-clinical and clinical development of these molecules should simplify the further development of protein therapeutics that follow . Specific studies with recombinant human interleukin-4 (rhuIL-4) were reviewed in detail as part of a pre-clinical safety evaluation . Native IL-4 has properties that exemplify many of the immune recognition-induced lymphokines and is produced principally by activated T-lymphocytes CD4+ . It is a co-factor in B-cell proliferation and enhances ex vivo B-cell expansion and is believed to be a candidate for the treatment of refractory cancer based on this immune enhancement ability . rhuIL-4 is a 15,400 molecular weight cytokine produced in a yeast expression system.(ABSTRACT TRUNCATED AT 250 WORDS) Inflammation, 1992 Apr, 16(2), 83 - 91 Phagocytosis by lipopolysaccharide-primed human neutrophils is associated with increased extracellular release of reactive oxygen metabolites; Follin P et al.; The effect of priming human neutrophils with lipopolysaccharide was investigated regarding the respiratory burst activity generated during phagocytosis of IgG- or C3b-opsonized yeast particles . LPS pretreatment significantly enhanced the respiratory burst activity, measured as luminol-amplified chemiluminescence, of both types of opsonized particles . In control cells most of the activity was produced intracellularly, probably in the phagosomes . In the primed cells, however, extracellular release of reactive oxygen metabolites was significantly increased during Fc- and CR3-mediated phagocytosis (P less than 0.01 and P less than 0.002, respectively) . The release was most pronounced when using C3b-opsonized particles . Potent oxygen metabolites acting together with lysosomal enzymes are of importance in inflammatory-induced tissue damage . An increased extracellular release of reactive oxygen species by phagocytizing primed neutrophils can therefore lead to greater damage to the surrounding tissues. Comput Appl Biosci, 1992 Apr, 8(2), 177 - 84 DNA Translator and Aligner: HyperCard utilities to aid phylogenetic analysis of molecules; Eernisse DJ; DNA Translator and Aligner are molecular phylogenetics HyperCard stacks for Macintosh computers . They manipulate sequence data to provide graphical gene mapping, conversions, translations and manual multiple-sequence alignment editing . DNA Translator is able to convert documented GenBank or EMBL documented sequences into linearized, rescalable gene maps whose gene sequences are extractable by clicking on the corresponding map button or by selection from a scrolling list . Provided gene maps, complete with extractable sequences, consist of nine metazoan, one yeast, and one ciliate mitochondrial DNAs and three green plant chloroplast DNAs . Single or multiple sequences can be manipulated to aid in phylogenetic analysis . Sequences can be translated between nucleic acids and proteins in either direction with flexible support of alternate genetic codes and ambiguous nucleotide symbols . Multiple aligned sequence output from diverse sources can be converted to Nexus, Hennig86 or PHYLIP format for subsequent phylogenetic analysis . Input or output alignments can be examined with Aligner, a convenient accessory stack included in the DNA Translator package . Aligner is an editor for the manual alignment of up to 100 sequences that toggles between display of matched characters and normal unmatched sequences . DNA Translator also generates graphic displays of amino acid coding and codon usage frequency relative to all other, or only synonymous, codons for approximately 70 select organism-organelle combinations . Codon usage data is compatible with spreadsheet or UWGCG formats for incorporation of additional molecules of interest . The complete package is available via anonymous ftp and is free for non-commercial uses. J Interferon Res, 1992 Apr, 12(2), 119 - 29 Molecular cloning of porcine Mx cDNAs: new members of a family of interferon-inducible proteins with homology to GTP-binding proteins; Muller M et al.; Porcine cells treated with interferon (IFN) or double-stranded RNA synthesize two proteins that exhibit high homology of the amino acid sequence to mouse Mx1 protein involved in selective resistance to influenza virus . A full-length cDNA clone (poMx1) encoding the porcine Mx1 protein was isolated and sequenced . It contained an open reading frame of 663 amino acids . The predicted molecular weight of 75.6 kD is in good agreement with the apparent molecular mass of the two immunoprecipitable proteins of 76 kD and 73 kD determined by SDS polyacrylamide gel electrophoresis . A second cDNA (poMx2) was characterized which was incomplete in the 5' region . A comparison of all known Mx proteins revealed an average homology of 67.5% . The porcine Mx1 polypeptide is most closely related to human MxA (p78), murine Mx2, rat Mx2, and rat Mx3 proteins . The amino-terminal halves of all Mx proteins are highly conserved and possess three consensus elements in proper spacing, characteristic of GTP-binding domains . The Mx family shows in their amino termini striking homology to previously characterized Mx-related proteins playing roles in the intracellular vectorial transport of proteins--the products of the yeast Vps1 locus and the dynamins. Carcinogenesis, 1992 Apr, 13(4), 609 - 15 Reversion of the hprt mutant clone SP5 by intrachromosomal recombination; Zhang LH et al.; The spontaneous hprt mutant clone SP5, derived from V79 Chinese hamster cells, was shown to exhibit a duplication of approximately 2 kb, including exon 2 and its flanking intron sequences, inserted into the intron 1 sequence of the hprt gene . The most striking feature of SP5 is that this clone is quite unstable, demonstrating an extremely high spontaneous reversion frequency . Molecular analysis of 25 independent revertant clones of SP5 indicated that they arose after precise deletion of the duplicated fragment in the hprt gene . Reversion of SP5 could be induced by agents which damage DNA by different mechanisms, but there was no correlation with induction of the forward mutations . Based on these results, we suggest that intrachromosomal recombination must be responsible for the spontaneous reversion of SP5 . Genetic recombination in somatic cells has been suggested to be involved in the multistep process of carcinogenesis . Since the ability to induce intrachromosomal recombination in yeast has been shown to be highly correlated with non-mutagenic as well as mutagenic carcinogens, it is of great interest to investigate similar systems in mammalian cells . The SP5 cell line may be unique for such a purpose, since this mutant clone contains an endogenic marker for studying the process of intrachromosomal recombination. Eur J Biochem, 1992 Apr 1, 205(1), 211 - 6 Molecular characterization of a Leishmania donovani infantum antigen identified as histone H2A; Soto M et al.; A Leishmania donovani infantum promastigote cDNA expression library was screened with a serum obtained from a dog naturally infected with this parasite . One of the positive clones obtained revealed nucleotide sequence similarities with the histone H2A genes from various organisms . Northern blot analyses and sequence data of three independently isolated cDNA clones indicated that the Leishmania H2A mRNAs are polyadenylated, as are the basal histone mRNAs of higher eukaryotes and the histone mRNAs of yeast . The analysis of the genomic distribution of the DNA coding for histone H2A suggested that, in L . d . infantum, there are at least four genes coding for the H2A protein . It is likely that there is a simultaneous expression of at least two of the H2A genes since differences in nucleotide sequence between two of the sequenced cDNAs were observed . Affinity-purified antibodies against the beta-galactosidase-fused H2A protein recognize specifically a Leishmania protein band with a molecular mass of 14 kDa. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1992 Apr, 14(2), 155 - 8 {An approach to the preparation of digestive enzyme-drug for oral administration: enteric-soluble disaccharidase microcapsules}; Wang X; For the purpose of replacement therapy in cases of disaccharide intolerances, yeast disaccharidases were prepared as enteric-soluble microcapsules to prevent their inactivation by gastric juice and alimentary proteases . The enteric-soluble microcapsules have a mean diameter of 0.544 +/- 0.058 mm . Following encapsulation, activity recoveries for invertase and lactase were 84.4 +/- 10.6% and 70.1 +/- 5.5%, respectively . Over 55% of the remaining microcapsulated disaccharidase activity was retained in artificial gastric juice during 2 h of coincubation . Over 90% of the enzyme activity was released from the microcapsules within 1 h in artificial intestinal juice. Curr Opin Cell Biol, 1992 Apr, 4(2), 144 - 8 Cell proliferation and control; Pines J; In the past year much of the focus in cell-cycle research has turned from the regulation of mitosis to the control of the initiation of DNA replication . Novel findings include the discovery of vertebrate G1 cyclins, an additional cdc2-related kinase potentially involved in G1 phase, and a positive-feedback loop regulating the start of the cell cycle in yeast. Lab Invest, 1992 Apr, 66(4), 498 - 503 Monocyte chemoattractant protein 1 in a rat model of pulmonary granulomatosis; Jones ML et al.; Monocyte chemoattractant protein 1 (MCP1), also known as monocyte chemotactic and activating factor, possesses potent chemotactic activity for monocytes and can augment monocyte tumoristatic activity against some tumor cell lines . While these activities suggest a role in inflammatory and immunologic processes, the biologic role of MCP1 has not been studied in vivo . Glucan-induced pulmonary granulomatosis in the rat is an ideal model in which to study the role of MCP1 because the granulomas are monocyte/macrophage rich . Intravenous infusion of particulate yeast cell wall glucan resulted in the synchronous development of angiocentric pulmonary granulomas . Early lesions (6 hours) were characterized by intravascular glucan aggregates surrounded by neutrophils and foci of alveolar hemorrhage while later appearing granulomatous lesions (48 to 96 hours) were dominated by monocytes and macrophages . Granuloma formation was paralleled by a peripheral blood monocytosis . Analysis of bronchoalveolar lavage (BAL) fluid revealed an early, transient rise in tumor necrosis factor activity followed by a marked rise in monocyte-specific chemotactic activity . The rise in BAL fluid monocyte chemotactic activity, which coincided with the development of the monocyte/macrophage-rich granulomas, was preceded by a marked increase in whole lung MCP1 mRNA expression . BAL fluid monocyte chemotactic activity could be nearly completely neutralized with antibody directed against rat MCP1 . These studies demonstrate that MCP1 mRNA expression is upregulated in glucan-induced pulmonary granulomatosis and that MCP1 is present in BAL fluid . Intrapulmonary granulomatosis may be important in the pathogenesis of granuloma formation. J Clin Invest, 1992 Apr, 89(4), 1172 - 7 Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core . Implications for paroxysmal nocturnal hemoglobinuria; Thomas LJ et al.; A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized . The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level . We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level . The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway . Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants . These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences . Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria. Mol Biol Cell, 1992 Apr, 3(4), 403 - 14 SpCoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury; Smith LC et al.; SpCoel1 is a single copy gene that is specifically expressed in most of the coelomocytes of the adult purple sea urchin, Strongylocentrotus purpuratus . The 4-kb transcript from this gene has a relatively short (426 nucleotide) open reading frame (ORF) with long 3' and 5' untranslated regions . The ORF encodes a protein that has strong amino acid sequence similarity to profilins from yeast to mammals . Transcript titrations of SpCoel1 show significant increases per coelomocyte in animals that have been physiologically challenged . Increases in transcript levels are of similar magnitudes between animals receiving different treatments, such as injuries from needle punctures or from injections of foreign cells . The evidence presented here implies a molecular mechanism by which this lower deuterostome defense system responds to external insult, viz that an external "injury signal" activates a signal transduction system, which in turn mediates the alterations in cytoskeletal state that are required for coelomocyte activation. Mycopathologia, 1992 Apr, 118(1), 29 - 36 Use of a colorimetric system to detect enzymes expressed by germinating conidia of entomopathogenic fungi; el-Sayed GN et al.; An apiZYM system, with 19 substrates, was used to detect enzymes expressed by germinating conidia of Nomuraea rileyi (5 isolates), Nomuraea atypicola, Nomuraea anemonoides, Beauveria bassiana and Metarhizium anisopliae . Similar enzyme profiles were obtained for two of the N . rileyi isolates (Mississippi, Ecuador) regardless of whether culture medium (Sabouraud-maltose-yeast) or cuticle (from larvae of Trichoplusia ni, Heliothis zea or Heliothis virescens) were used as substrates . Centroid-clustering analysis revealed three distinct enzyme profiles. Biol Trace Elem Res, 1992 Apr-Jun, 33, 197 - 204 Selenium content and distribution in rat tissues irradiated with gamma rays; Djujic IS et al.; The effects of supplementation with selenous yeast and ionizing radiation on selenium (Se) content and distribution were evaluated in rat tissues (liver, kidney, spleen, heart, muscle, blood, front brain, hind brain, hypothalamus, pituitary, adrenal glands, testes, and hair) . This study had 16 Se-supplemented (0.5 micrograms Se/d) and 16 placebo adult male Wistar rats . One half of the animals (eight Se-supplemented and eight placebos) were irradiated with a single dose of 4.2 Gy from a Co-60 source and sacrificed 7 d after irradiation along with nonirradiated animals and analyzed for Se content determination . The data obtained showed that selenous yeast supplementation increased Se levels in rat tissues (highest increases in hypothalamus, 161%; hind brain, 126%; spleen, 110%; and adrenal gland, 105%) . Ionizing radiation induced significant changes in Se content and distribution (decrease in liver, blood, hair, femoral muscle, spleen, and hypothalamus; increase in kidney, testes, adrenal glands, and brain of placebo group) . Supplementation with selenous yeast reduces changes in Se content and distribution after irradiation . It seems that the animal tissue susceptibility to oxidative damage may be correlated to their ability to retain Se in tissues. Appl Microbiol Biotechnol, 1992 Apr, 37(1), 109 - 13 Comparison of the catalytic and inhibitory properties of Pachysolen tannophilus xylose reductase to rat lens aldose reductase; Davis RA et al.; The catalytic and inhibitory profiles of xylose reductase isolated from the yeast Pachysolen tannophilus (PTXR) are compared to those of aldose reductase (AR) obtained from rat lens . While both PTXR and rat lens AR are NADPH-specific enzymes and have an affinity for a variety of substrates such as D-xylose, D,L-glyceraldehyde, and 4-nitrobenzaldehyde, the enzymes differ in their substrate affinity profiles . Also, PTXR is not inhibited by standard inhibitors of AR thus supporting a hypothesis that this enzyme may not possess the inhibitor binding site found in rat lens AR. Hum Genet, 1992 Apr, 89(1), 33 - 6 Anonymous markers located on chromosome 6 in the HLA-A class I region: allelic distribution in genetic haemochromatosis; Boretto J et al.; Two yeast artificial chromosomes of the HLA class I region were subcloned . Four of the subclones studied displayed restriction polymorphisms that corresponded to six bi-allelic series . Allelic distribution of the anonymous markers was then studied by comparing a control population with a group of patients with familial haemochromatosis . Only one marker presents an unequivocal association with the haemochromatosis gene and is 100 kb centromeric to HLA-A . This association however is not as strong as with HLA-A3 . The results suggest two possible locations for the haemochromatosis gene: less than 100 kb centromeric to the HLA-A locus, or on the telomeric side. Glycoconj J, 1992 Apr, 9(2), 75 - 81 O-glycosidically linked oligosaccharides from peptidorhamnomannans of Sporothrix schenckii; Lopes-Alves LM et al.; beta-Elimination of peptidorhamnomannans purified from yeast-like and mycelial phases of Sporothrix schenckii released neutral and acidic reduced oligosaccharides that were O linked to serine and/or threonine . Man-(alpha 1-2)Man-ol, Rha(alpha 1-3)Man(alpha 1-2)Man-ol, Rha(alpha 1-4)GlcA(alpha 1-2)Man(alpha 1-2)Man-ol, and Rha(alpha 1-4){Rha(alpha 1-2)} GlcA(alpha 1-2)Man(alpha 1-2)Man-ol were characterized based on methylation analysis, proton magnetic resonance and fast atom bombardment mass spectrometry. Brain Pathol, 1992 Apr, 2(2), 121 - 32 Defects of mitochondrial DNA; Zeviani M et al.; In the past few years several syndromes have been associated with lesions of the human mitochondrial DNA . MtDNA is a small, circular extra-nuclear chromosome encoding essential components of the respiratory chain . MtDNA-related syndromes can be divided into two groups: mitochondrial encephalomyopathies, characterized by the presence of ragged-red fibres (RRF) as the morphological hallmark, or "pure" encephalopathies with no gross morphological abnormalities in muscle . The first group includes myoclonic epilepsy with ragged-red fibres (MERRF), mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Kearns-Sayre syndrome (KSS), chronic progressive external ophthalmoplegia (CPEO) and a new entity, maternally inherited myopathy and cardiomyopathy . The second group includes Leber's Hereditary Optic Neuroretinopathy (LHON) and the newly described ataxia-retinitis pigmentosa-dementia complex . Three kinds of molecular lesions have been identified: point mutations of protein encoding mtDNA-genes (similar to yeast mit- mutations); point mutations of mtDNA-tRNA genes (similar to yeast syn- mutations); and large-scale rearrangements of mtDNA (similar to yeast rho- mutations) . In general, "mit-" mutations are responsible for non-RRF encephalopathies, while "syn-" and "rho-" mutations are associated with mitochondrial encephalomyopathies with RRF . Furthermore, point mutations (mit- and syn-) are usually maternally- inherited, while large-scale mtDNA rearrangements are either sporadic or inherited as mendelian traits . In most cases, the molecular detection of the known defects of mtDNA can be carried out by non-invasive techniques, thus making it an easy and relatively inexpensive procedure in the differential diagnosis of the mitochondrial disorders, a rapidly expanding area of clinical neurology. Hum Mol Genet, 1992 Apr, 1(1), 3 - 6 Beginning or end? Telomere structure, genetics and biology; Kipling D et al.; The word telomere derives from the Greek word telos meaning 'end', roughly translating as 'the thing at the end' when the end is that of a chromosome . Telomeres belie their apparent simplicity of structure by being involved in a wide range of diverse biological phenomena . Much of our understanding of telomere behaviour comes from studies in lower eukaryotes such as ciliates and yeast, the subject of many recent reviews . Here we concentrate on the mammalian telomere, recent progress in its study, and how recent evidence for an involvement of telomeres in the regulation of gene expression and DNA replication in yeast points to new aspects of mammalian telomere function yet to be explored. Nature, 1992 Mar 26, 356(6367), 353 - 5 The wee1 protein kinase is required for radiation-induced mitotic delay; Rowley R et al.; Cellular feedback or 'checkpoint' mechanisms maintain the order of completion of essential, cell-cycle related functions . In the budding yeast, for example, the RAD9 gene product is required to delay progression into mitosis in response to DNA damage . Similarly, in fission yeast, the cdc25 and cdc2 gene products influence the ability of cells to delay mitosis in response to the inhibition of DNA synthesis . Because these two checkpoint controls regulate the same event, mitosis, we observed the effect of gamma-irradiation on cell cycle progression in fission yeast, to test whether the two controls require the same cell-cycle regulatory elements . We show that gamma-radiation-induced mitotic delay requires functional wee1 protein kinase but does not seem to involve the cdc25 pathway . Mitotic delay in response to DNA damage is thus distinct from the delay induced by inhibition of DNA synthesis, which involves cdc25 but is not dependent on wee1. Eur J Obstet Gynecol Reprod Biol, 1992 Mar 23, 44(1), 77 - 80 Vulvovaginal candidiasis refractory to treatment with fluconazole; Arilla MC et al.; We present the case of an infertile patient, whose first attempt at IVF had to be postponed for 18 months due to a vulvovaginal yeast infection refractory to treatment . The main causative organism was a Candida glabrata strain resistant to all the imidazolic agents tested . The organism and the host's humoral status were studied in depth, looking for possible causes of the refractoriness to treatment. J Mol Biol, 1992 Mar 20, 224(2), 343 - 58 Localization and DNA sequence of a replication origin in the rhodopsin gene locus of Chinese hamster cells; Gale JM et al.; A chromosomal origin of DNA replication has been localized within the single-copy rhodopsin gene locus in Chinese hamster (line CHO) cells using two methods . In the first method, single-copy segments were identified at 3 to 15 kb intervals within approximately 75 kb (kb = 10(3) bases) of cloned genomic DNA containing the early-replicating rhodopsin gene near its middle . The cloned single-copy segments were then used as hybridization probes to quantify the replication of their corresponding genomic segments as synchronized cells progressed into S phase . In the second method, genomic DNA synthesized in vivo or in permeabilized early S phase cells was hybridized with slot-blots of the cloned single-copy DNA segments to identify the earliest replicating part of the 75 kb mapped region . The first method indicates that the earliest replicating DNA is located within a 10 kb region beginning 4 kb upstream from and extending 1 kb beyond the rhodopsin gene . The second method confirms the location in the vicinity of the rhodopsin gene and indicates that the earliest replicating region is located within or very near the 4.5 kb rhodopsin gene itself . An extended region of 12 kb that encompasses the entire early-replicating region has been sequenced for analysis and comparison with currently characterized origin regions associated with the CHO dihydrofolate reductase (dhfr) and human c-myc genes . There are several sequence similarities between the dhfr rhodopsin origin regions, including common transcription promoter consensus sequences, rodent Alu repeats with their 3'-A+T rich flanking sequences, A+T-rich yeast ARS and Drosophila SAR consensus sequences, and simple (GA)n repeats, but there are no extended regions of direct similarity . The rhodopsin gene locus is the second sequenced CHO origin region. Biochem Pharmacol, 1992 Mar 17, 43(6), 1345 - 51 Mechanisms of inactivation of Schistosoma mansoni and mammalian glutathione S-transferase activity by the antischistosomal drug oltipraz; Nare B et al.; Glutathione S-transferase (GST) purified from Schistosoma mansoni or human placenta was inhibited by the antischistosomal drug oltipraz (OPZ) in a time- and concentration-dependent manner . Inhibition of placenta GST was complete at a low concentration of drug, whereas that of parasite GST was incomplete and relatively high amounts of OPZ were needed to reach 50% inhibition . Complete reactivation of GST from placenta was achieved with dithiothreitol (DTT) and other sulfhydryl-containing compounds, while the inactivation of parasite GST was irreversible . The oxy-derivative of OPZ (RP 36,642), in which the thione sulfur is replaced with oxygen, did not inhibit GST activity . There were no differences between OPZ and RP 36,642 in their patterns of binding to the hydrophobic non-substrate site of GST . GST from the placenta incorporated much higher levels of {14C}N-ethylmaleimide compared to schistosome GST . The incorporation of {14C}N-ethylmaleimide by GST was inhibited by OPZ but not by RP 36,642 . Yeast and S . mansoni hexokinases were similarly inhibited by OPZ but not by RP 36,642 . Both hexokinase preparations recovered their activity following incubation with DTT . These data suggest that the inactivation of these enzymes by OPZ is a result of its interaction with their SH groups . Thus, the antischistosomal activity of OPZ may be accounted for by its interaction with the SH groups of macromolecules in general. Carbohydr Res, 1992 Mar 16, 226(1), 79 - 89 Synthesis and enzyme-inhibitory activity of methyl acarviosin analogues having the alpha-manno configuration; Ogawa S et al.; Two methyl acarviosin analogues 3a and 4a, having the alpha-manno configuration, and their dihydro derivatives 6a and 7a were synthesised by coupling the protected pseudo-sugar epoxides with methyl 4-amino-4-deoxy- and -4,6-dideoxy-alpha-D-mannopyranoside . Similarly, two analogous compounds 5a and 8a composed of the 1,6-anhydro-beta-D-mannopyranose residues were prepared . Compound 7a showed mild inhibitory activity against Jack bean alpha-D-mannosidase, and 3a was a moderate inhibitor of both alpha-D-mannosidase and yeast alpha-D-glucosidase. J Immunol, 1992 Mar 15, 148(6), 1804 - 11 Regulation of synthesis of p34cdc2 and its homologues and their relationship to p110Rb phosphorylation during cell cycle progression of normal human T cells; Lucas JJ et al.; In yeast, the protein kinase p34cdc2 plays a role in regulating both the G2 to M and G1 to S phase transitions . The discovery of multiple homologues of the protein in cells of higher eukaryotic organisms suggests that different cell cycle regulatory events may be performed by different kinases in such cells . Here, the synthesis and metabolism of the human forms of these proteins are described in a normal human cell type, peripheral blood T lymphocytes that have been stimulated to enter the cell cycle in vitro . Using a carboxyl-terminus antiserum specific for true p34cdc2, the protein could first be found in T cells at about 24 to 30 h after stimulation, just before the initiation of DNA synthesis . Three forms of the enzyme could be resolved by denaturing gel electrophoresis: an unphosphorylated form with an apparent molecular mass of 34,500 daltons and two phosphorylated derivatives . In cells synchronized at G2/M phase with nocodazole, p34 was almost entirely in the unphosphorylated form whereas the phosphorylated derivatives were more predominant in cultures arrested at the G1/S border with aphidicolin . The relationship of p34 synthesis to the phosphorylation of p110Rb, an event known to be associated with passage through late G1 and/or the G1/S phase transition, was also investigated . It was noted that p110Rb phosphorylation began before p34 synthesis first became detectable . Furthermore, it appeared that the two events could be largely uncoupled by treating cells with deferoxamine (10 microM), an iron chelating agent that arrests T cells at a point in late G1 phase but substantially before the G1 to S phase transition . Under these conditions, p110Rb phosphorylation was almost completely accomplished in the absence of significant p34 synthesis, a finding that suggests that most or all of p110 phosphorylation is performed by kinases other than p34 . Because of this observation, extracts were next examined for p34-like molecules using an antibody against the so-called PSTAIRE domain found in all cdc2 homologues identified to date . A species of protein with a mobility slightly less than true p34 was found, even in resting T cells . Upon stimulation, this protein increased slightly in amount, and a second protein with a mobility greater than p34, a putative p33cdk2, was seen . Not only was the appearance of these proteins not inhibited by deferoxamine but they accumulated in cultures treated with the drug, suggesting that p33, and not p34, may be the G1 phase kinase for p110Rb.(ABSTRACT TRUNCATED AT 400 WORDS) Gene, 1992 Mar 15, 112(2), 147 - 55 Functional cloning vectors for use in directional cDNA cloning using cohesive ends produced with T4 DNA polymerase; Kuijper JL et al.; This paper describes the construction of 'Prime' cloning vectors, which include phage lambda and plasmid vectors useful for functional cloning in oocytes, yeast, and mammalian cells, and their use in a 'Prime' cloning system . The system takes advantage of the very active and precise 3' exonuclease activity of T4 DNA polymerase to produce single-stranded (ss) ends (cut-back) of vector and insert DNA . This results in the highly efficient directional cloning of cDNA and PCR-amplified DNA . The system obviates the need to digest insert DNA with a restriction endonuclease to unveil cloning sites, and thus eliminates the chance of internal digestion of the insert DNA . The cloning of PCR-amplified DNA, which is sometimes difficult, is made routine with this system . The 'Prime' sequence is included in vector cloning sites and cDNA and PCR primers . The 'Prime' sequence was chosen so that the ss sticky ends are nonpalindromic and will hybridize only to the appropriate partners . This makes cloning with the 'Prime' system very efficient, because neither the vector nor insert DNA is lost to unproductive self-hybridization. J Biol Chem, 1992 Mar 15, 267(8), 5676 - 9 Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis; Imamura T et al.; We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1) . The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1 . In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells . Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus . Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants . These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro. Science, 1992 Mar 13, 255(5050), 1404 - 8 Participation of the intron in the reaction catalyzed by the Xenopus tRNA splicing endonuclease; Baldi MI et al.; Introns have generally been assumed to be passive in the transfer RNA splicing reaction . Experiments have now been done showing that the endonuclease is able to cut a precursor provided that a base in the single-stranded loop of the intron can pair with the base of the 5' exon situated at the position that immediately follows the anticodon stem (position 33 in the yeast tRNA isoacceptor pre-tRNA(Leu)3, position 32 in yeast pre-tRNA(Phe)) . The elucidation of the role of the intron reveals that in addition to the conserved bases, there are positions in the mature domain which, although not necessarily occupied by the same base in all pre-tRNA's, nevertheless have a fundamental role in the splicing reaction . These positions are termed cardinal positions. Nature, 1992 Mar 12, 356(6365), 159 - 61 Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits; Federman AD et al.; Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits . In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively . By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways . Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma . This effect of beta gamma requires costimulation by alpha s-GTP . This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active . We now confirm this prediction . Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis . Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation . These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II. J Virol, 1992 Mar, 66(3), 1389 - 93 LRV1 viral particles in Leishmania guyanensis contain double-stranded or single-stranded RNA; Weeks R et al.; The 32-nm-diameter spherical viral particles found in the cytoplasm of Leishmania guyanensis CUMC1-1A sediment at 130S and have a buoyant density of approximately 1.4 g/ml in cesium chloride gradients . These particles contain a 5.3-kb double-stranded RNA, while single-stranded RNA that corresponds to the viral positive strand is associated with less-dense particles . These results suggest a conservative and sequential mode of LRV1 viral RNA replication that is exemplified by the ScV L-A virus of yeast. Nucleic Acids Res, 1992 Mar 11, 20(5), 1087 - 91 Evolutionary variation of the CCAAT-binding transcription factor NF-Y; Li XY et al.; NF-Y is a CCAAT-specific transcription factor thought to be involved in the regulation of a variety of eukaryotic genes . It shows a striking sequence similarity with the yeast factor HAP2/3 . In an attempt to trace back its evolutionary history, we succeeded in isolating NF-Y cDNA clones from a plant and from several species of vertebrates . The patterns of sequence conservation delineate potential functional domains: A central, highly conserved, domain likely responsible for DNA-binding and subunit interaction; more evolutionarily flexible flanking regions, in which variability is clustered, individualizing conserved glutamine or acidic amino-acids putatively involved in protein-protein contacts. Biochim Biophys Acta, 1992 Mar 5, 1116(1), 67 - 71 Formation of beta-galactosyl compounds of pyridoxine in growing culture of Sporobolomyces singularis; Suzuki Y et al.; Several pyridoxine compounds were found to be formed in a high yield in a growing culture of Sporobolomyces singularis containing lactose and pyridoxine . Three compounds, I, II and III, were isolated from cultured broth by Dowex 50W-X8 column chromatography, paper chromatography, lyophilization, and then obtained as needle crystals (m.p . (decomp.): I, 204-206 degrees C; II, 192-194 degrees C; III, 222-223 degrees C . {alpha}D16: I, -27.7 degrees (c = 3.82, H2O); II, -1.6 degrees (c = 2.52, H2O); III, +8.3 degrees (c = 3.98, H2O)) . Compounds I, II and III were identified as 5'-O-(beta-D-galactopyranosyl)-pyridoxine, 4'-O-(beta-D-galactopyranosyl)-pyridoxine, and 4'-O-(beta-D-galactopyranosyl-(1----4)-beta-D-galactopyranosyl)-pyrid oxi ne, respectively, on the basis of the various experimental results, viz., ultraviolet, infra-red, 1H-NMR, and 13C-NMR spectra, products by hydrolysis with acid and with alpha- and beta-galactosidases, migration on paper electrophoresis, and Gibbs reaction in the presence and absence of boric acid . Also, the yeast produced a remarkable amount of beta-glucosyl compounds of pyridoxine in cultured broth, when grown on cellobiose and pyridoxine. Nature, 1992 Mar 5, 356(6364), 80 - 3 Gamma-tubulin is a centrosomal protein required for cell cycle-dependent microtubule nucleation; Joshi HC et al.; gamma-Tubulin is a newly identified member of the tubulin family whose sequence is highly conserved from yeast to man . This minor microtubule protein is localized to the microtubule organizing centres and a mutation in the gene encoding it produces a microtubuleless mitotic arrest in the filamentous fungus Aspergillus nidulans . Here we investigate the in vivo function of gamma-tubulin in mammalian cells using a synthetic peptide to generate a polyclonal antibody that binds to a highly conserved segment of gamma-tubulin . After microinjection into cultured mammalian cells, immunofluorescence localization revealed that this antibody binds to native centrosomes at all phases of the cell cycle . In the presence of the gamma-tubulin antibody, microtubules fail to regrow into cytoplasmic arrays after depolymerization induced by nocodazole or cold . Furthermore, cells injected immediately before or during mitosis fail to assemble a functional spindle . Thus in vivo gamma-tubulin is required for microtubule nucleation throughout the mammalian cell cycle. Eur J Epidemiol, 1992 Mar, 8(2), 305 - 8 Hansenula anomala fungemia in an infant with gastric and cardiac complications with a review of the literature; Sekhon AS et al.; A 6-month-old female infant, with a birth weight of 2.74 kilograms, was born with multiple congenital abnormalities, including gastric and gastrointestinal defects . She was admitted to the hospital with hematemesis . The patient could not be fed orally, and parenteral nutrition was initiated through a central venous catheter . Following pyloroplasty, she developed superior vena cava syndrome, renal disfunction and episodes of sepsis . Stool and respiratory specimens were negative for fungi, but four blood cultures yielded Hansenula anomala var . anomala . Cultures for fungi from intravenous catheter tips were negative . The baby was treated with amphotericin B (am B) and 5-fluorocytosine (5-FC), (amB; 0.1 mg/kg body weight and 5-FC, 100 mg, q.i.d.) . The minimal inhibitory concentrations of am B, 5-FC, am B + 5-FC (1:1, w:w) and fluconazole to H . anomala were 1.56, less than 0.195, 1.56, and 1.56 micrograms, respectively . Following antifungal therapy and removal of the catheter, the patient tolerated oral feeding and, at the time of discharge, her weight had increased to 4.91 kg . This report records H . anomala as an opportunistic yeast pathogen for the first time in Alberta, Canada . Previously published cases of H . anomala infections are reviewed. Mycopathologia, 1992 Mar, 117(3), 139 - 44 Detection of cellular immunity with the soluble antigen of the fungus Sporothrix schenckii in the systemic form of the disease; Carlos IZ et al.; Sporothrix schenckii is the etiologic agent of sporotrichosis, a mycosis of world-wide distribution more commonly occurring in tropical regions . The immunological mechanisms involved in the prevention and control of sporotrichosis are not fully understood but apparently include both the humoral and cellular responses . In the present investigation, cellular immunity was evaluated by in vivo and in vitro tests in mice infected with yeast-like forms of S . schenckii . The disease developed systemically and cellular immunity was evaluated for a period of 10 weeks . The soluble antigen utilized in the tests was prepared from yeast form of the fungus through the sonication (20 min: 10 sonications at 50 W at 2-min intervals) . Delayed hypersensitivity and lymphocyte transformation tests showed that the cellular immune response was depressed between the 4th and 6th week of infection when the animals were challenged with the soluble fungal antigen . This depression frequently indicates worsening of the disease, with greater involvement of the host . This is a promising field of research for a better understanding of the pathogeny of this mycosis. Development, 1992 Mar, 114(3), 681 - 7 Inducible cell ablation in Drosophila by cold-sensitive ricin A chain; Moffat KG et al.; We have developed a system for temperature-inducible killing of specific cells in the fruitfly Drosophila melanogaster . The system overcomes many of the limitations of existing cell ablation methods and is in principle applicable to any non-homeothermic eukaryote . Temperature-sensitive and cold-sensitive mutations in the ricin toxin A chain (RTA) of castor bean were generated in yeast . One cold-sensitive mutation, RAcs2, produced temperature-dependent ablation of eye cells in Drosophila when expressed under control of the eye-specific sev enhancer . At 29 degrees C, cell death was observed within 7 hours in the developing eye and no obvious toxic effects were observed elsewhere; at 18 degrees C, extremely low toxicity was observed . DNA sequencing of RAcs2 revealed a single amino acid substitution in the RTA active site cleft. J Biochem (Tokyo), 1992 Mar, 111(3), 296 - 301 Molecular and enzymatic properties of furin, a Kex2-like endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites; Hatsuzawa K et al.; We have recently shown that furin, a mammalian homologue of the yeast precursor-processing endoprotease Kex2, is involved in precursor cleavage at sites marked by the Arg-X-Lys/Arg-Arg motif within the constitutive secretory pathway . In this study, we analyzed molecular and enzymatic properties of furin expressed in Chinese hamster ovary cells using gene transfer techniques . COOH-terminal truncation analyses indicate that the polypeptide region significantly conserved among the Kex2 family members is required for the endoprotease activity of furin, while the COOH-terminal unconserved region containing the Cys-rich domain and the transmembrane domain is dispensable . A mutant of furin truncated up to the transmembrane domain from the COOH-terminus was secreted into the culture medium as an active form . The sequence requirements for precursor cleavage of this truncated furin determined in vitro were similar to those of wild-type furin determined by expression studies in cultured cells . It had a strong resemblance to the Kex2 protease in the inhibitor profile and pH dependency . These observations support the notion that furin is the endogenous endoprotease involved in precursor cleavage at Arg-X-Lys/Arg-Arg sites. Bioessays, 1992 Mar, 14(3), 151 - 60 Sorting of proteins to the vacuoles of plant cells; Vitale A et al.; The secretory system of plant cells sorts a large number of soluble proteins that either are secreted or accumulate in vacuoles . Secretion is a bulk-flow process that requires no information beyond the presence of a signal peptide necessary to enter the endoplasmic reticulum . Many vacuolar proteins are glycoproteins and the glycans are often modified as the proteins pass through the Golgi complex . Vacuolar targeting information is not contained in glycans as it is in animal cells; rather, targeting information is in polypeptide domains as it is in yeast cells . Several such domains have now been identified, but these show little or no amino acid sequence homology . We discuss the possibilities that targeting of protein to plant vacuoles may involve receptors as well as aggregation of protein at low pH. Plant Mol Biol, 1992 Mar, 18(5), 909 - 19 Cytoplasmic ribosomal protein S15a from Brassica napus: molecular cloning and developmental expression in mitotically active tissues; Bonham-Smith PC et al.; We have isolated two cDNA clones which appear to encode the 40S ribosomal subunit protein S15a from Brassica napus (oilseed rape) . The open reading frame in both clones contains 390 bases, encoding a deduced polypeptide sequence of 130 amino acids (100% homology between clones) with 76% sequence identity to the N-terminal 37 amino acids of the rat ribosomal protein S15a and 80% identity to the S24 polypeptide of yeast . Both the yeast and rapeseed proteins have a net positive charge of +9 and the rapeseed S15a protein has a molecular mass of 14778 Da compared to 14762 Da for the yeast protein . The rapeseed ribosomal protein S15a is encoded by a small multi-gene family with at least two actively transcribed members . A single transcript of ca . 1.0 kb, corresponding to ribosomal protein S15a, is abundant in actively dividing tissues such as apical meristem, flower buds and young leaves and less abundant in mature stem and fully expanded leaves. New Biol, 1992 Mar, 4(3), 173 - 87 Proteasomes: protein and gene structures; Tanaka K et al.; Proteasomes are ring- or cylinder-shaped particles that have a sedimentation coefficient of 20S and are composed of a characteristic set of small polypeptides . These particles have a latent multicatalytic proteinase activity . Recently, proteasomes were found to combine reversibly with multiple protein components to form 26S proteolytic complexes that catalyze ATP-dependent, selective breakdown of proteins ligated with ubiquitin . This suggests that the 26S complexes are a new type of ATP-requiring protease in eukaryotic cells . We have studied the structures of various eukaryotic proteasomes at the molecular level by physicochemical and recombinant DNA techniques and have proposed that the gross structures of proteasomes, such as their size and shape, have been highly conserved during evolution . Proteasome subunits appear to be encoded by a family of homologous genes named the "proteasome gene family," which may have evolved from a common ancestral gene . Evidence obtained by genetic analyses in yeast and studies on the levels of proteasome expression in various eukaryotic cells indicates that proteasomes have essential roles in the cell . In this review, we summarize available information on the protein and gene structures of proteasomes and discuss the biological functions of proteasomes. Clin Infect Dis, 1992 Mar, 14 Suppl 1, S30 - 6 Virulence properties and nonimmune pathogenetic mechanisms of fungi; Vartivarian SE; This article summarizes some of the potential fungal virulence factors, their effect on the host's defense systems, and their regulation by host factors . Immunopathogenesis is not discussed, but the role of adherence by fungal organisms to human surfaces and foreign bodies in pathogenesis is described . Dimorphism and, less commonly, phenotypic switching may play important roles in initiating and establishing infections by several fungi . Toxins, especially exotoxins, do not seem to participate significantly in pathogenesis; however, various enzymes (proteases, phospholipases) may represent virulence properties of Candida, Aspergillus, and a number of other fungi . The interaction of the organisms with their hormonal milieu, the iron-scavenging capacities of various fungi, and their potential role in pathogenesis are delineated . The immunosuppressive effects of certain fungal antigens, such as yeast mannans, are discussed. Clin Infect Dis, 1992 Mar, 14 Suppl 1, S148 - 53 Pathogenesis and treatment of recurrent vulvovaginal candidiasis; Sobel JD; In contrast to women who experience infrequent episodes of candidal vaginitis, patients with chronic and recurrent candidal vaginitis rarely have recognizable precipitating or causal factors . Analysis of vaginal yeast isolated from women with recurrent candidal vaginitis uncommonly reveals a higher percentage of non-albicans Candida species . There is no indication that resistance to azoles is a causal factor, and no other fungal virulence factors have been identified to explain the repeated attacks . Strain typing of sequential clinical isolates by means of molecular techniques indicate a pattern of relapse due to persistent yeast in the vagina rather than frequent vaginal reinfection . Attempts to reduce the number of attacks by treating sexual partners and suppressing a gastrointestinal tract focus have failed . Recent immunological studies suggest the possibility that an acquired Candida antigen-specific immunological deficiency results in uncontrolled vaginal Candida proliferation and hence repeated clinically evident attacks . Although no definitive cure for recurrent candidal vaginitis exists, numerous therapeutic maintenance regimens with azoles are available that effectively control symptomatic infection. Mol Biol Evol, 1992 Mar, 9(2), 278 - 84 Two ascomycete classes based on fruiting-body characters and ribosomal DNA sequence; Berbee ML et al.; Traditional fruiting body-based classification of ascomycetes has been under attack for 2 decades . Fruiting-body types can converge, and few researchers now assume that either the closed fruiting bodies (cleistothecia) characterizing the class Plectomycetes or the flask-shaped fruiting bodies (perithecia) characterizing the class Pyrenomycetes are stable, unifying characters . Unless we identify characters uniting major ascomycete groups, orders of ascomycetes remain narrowly defined, and supraordinal classification is impossible . We sequenced both strands of 18s rDNA from nine ascomycete fungi, adding three sequences from GenBank into our analysis . The phylogeny, inferred from 162 informative sites in 1,700 bp of DNA sequence data and using yeast as an outgroup, divided the fungi into two groups correlating well both with fruiting-body type and with the traditional classes Plectomycetes and Pyrenomycetes . Each group received strong statistical support . Genera producing cleistothecia, such as Talaromyces (with a Penicillium asexual state) and the human pathogen Ajellomyces capsulatus (causing histoplasmosis), fall within the plectomycete group . Plectomycetes also includes Eremascus albus and the bee pathogen Ascosphaera apis, although both lack typical fruiting bodies . The Dutch elm disease fungus groups with pyrenomycetes such as Neurospora, in spite of its confusing mixture of class-level characters. Genomics, 1992 Mar, 12(3), 581 - 9 The identification and characterization of KRAB-domain-containing zinc finger proteins; Constantinou-Deltas CD et al.; The zinc finger motif is a highly conserved tandemly repeated sequence of 28-30 amino acids that was first identified in transcription factor TFIIIA from Xenopus laevis . Subsequently, similar motifs were found and characterized in many genes from mammalian genomes and the genomes of lower eukaryotes such as Drosophila and yeast, thereby defining a large superfamily of genes . Non-finger-coding modules conserved among members of subfamilies of zinc finger genes have been described in the murine genome (finger-associated boxes, or FAX domain) and the human genome (Kruppel-associated boxes, or KRAB domain) . Here we report the identification and partial characterization of more members of the human KRAB-containing subfamily of genes . Based on Southern blot hybridization experiments, they also are zinc-finger-coding genes . All members share a highly homologous 42-amino-acid-long A element of the described KRAB domain . The conservation extends to the murine developmentally expressed zinc finger gene, mKr2 . The homologous sequences, however, are part of the 5'-untranslated region . In all cases for which there is adequate information, the KRAB domain is found at the NH2-terminus of the respective protein . In one zinc-finger-encoding cDNA clone that we characterized further in this work, BRc1744 (ZNF45), the KRAB domain most probably constitutes the entire second exon of the gene . Based on the data, it is tempting to speculate that the FAX- and KRAB-containing zinc finger genes define subfamilies of genes with overlapping functions that participate in the regulation of common or similar developmental programs. Oncogene, 1992 Mar, 7(3), 589 - 96 The Myb DNA-binding domain is highly conserved in Dictyostelium discoideum; Stober-Grasser U et al.; The c-myb proto-oncogene encodes a protein that is highly conserved among birds and mammals . The amino-terminal domain of c-Myb contains three imperfect tandem repeats of approximately 50 amino acids each . This domain is required for DNA binding and has also been conserved to varying degrees in invertebrates, plants and yeast . Given that myb-related genes appear to control cellular differentiation in a variety of eucaryotic systems, the presence of a myb gene in the cellular slime mold Dictyostelium discoideum might provide a tractable system for studying the role of myb in differentiation . Degenerate oligonucleotide primers encoding regions that are highly conserved in the vertebrate and Drosophila Myb DNA-binding domains were used to amplify a related domain from Dictyostelium genomic DNA, which was then used to isolate a genomic clone . The putative DNA-binding domain of Dictyostelium Myb is as closely related to vertebrate c-Myb as is Drosophila Myb (65% identity), whereas the known Myb-related proteins of plants and yeast are more distantly related . The conserved domain of Dictyostelium Myb is capable of binding to the same DNA sequence as the vertebrate and Drosophila Myb proteins . The remainder of the deduced amino acid sequence of Dictyostelium Myb shows no homology to the divergent domains of the known animal, plant and yeast Myb-related proteins . Evolutionary analysis implies that the duplications that generated the repeats of the Myb DNA-binding domain began prior to the divergence of animals, plants, cellular slime molds and yeast. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1875 - 9 The 65-kDa subunit of human NF-kappa B functions as a potent transcriptional activator and a target for v-Rel-mediated repression; Ballard DW et al.; Molecular cloning of the polypeptide component of the Rel-related human p75 nucleoprotein complex has revealed its identity with the 65-kDa (p65) subunit of NF-kappa B . Functional analyses of chimeric proteins composed of NF-kappa B p65 C-terminal sequences linked to the DNA-binding domain of the yeast GAL4 polypeptide have indicated that the final 101 amino acids of NF-kappa B p65 comprise a potent transcriptional activation domain . Transient transfection of human T cells with an expression vector encoding NF-kappa B p65, but not NF-kappa B p50, produced marked transcriptional activation of a basal promoter containing duplicated kappa B enhancer motifs from the long terminal repeat of type 1 human immunodeficiency virus . These stimulatory effects of NF-kappa B p65 were synergistically enhanced by coexpression of NF-kappa B p50 but were completely inhibited by coexpression of the v-rel oncogene product . Together, these functional studies demonstrate that NF-kappa B p65 is a transactivating subunit of the heterodimeric NF-kappa B complex and serves as one cellular target for v-Rel-mediated transcriptional repression. Proc Natl Acad Sci U S A, 1992 Mar 1, 89(5), 1641 - 5 Catalytic subunits of Aplysia neuronal cAMP-dependent protein kinase with two different N termini; Beushausen S et al.; Previously, two forms of cAMP-dependent protein kinase catalytic subunit generated by mutually exclusive use of two internal exon cassettes (A1 and A2) were demonstrated in Aplysia neurons . Here, it is shown that there also exist catalytic subunits with alternative N termini derived from two exons, N1 and N2, expressed in combination with either of the internal cassettes . Processed transcripts including N1 or N2 sequences are of about equal abundance in the nervous system, arise through alternative promoter use, and encode catalytically active polypeptides . The N2 amino acid sequence is 21 residues longer than the N1 sequence and is homologous to the nonmyristoylated N terminus of the TPK1 gene product, a yeast catalytic subunit homolog . These data support the view that cAMP-dependent protein kinase activity in Aplysia neurons is produced by a complex array of regulatory and catalytic subunits that generate multiple holoenzymes with a spectrum of properties. Eur J Biochem, 1992 Mar 1, 204(2), 865 - 73 Isolation, characterization, and sequence analysis of a cDNA clone encoding L-protein, the dihydrolipoamide dehydrogenase component of the glycine cleavage system from pea-leaf mitochondria; Bourguignon J et al.; L-protein is the dihydrolipoamide dehydrogenase component of the glycine decarboxylase complex which catalyses, with serine hydroxymethyltransferase, the mitochondrial step of photorespiration . We have isolated and characterized a cDNA from a lambda gt11 pea library encoding the complete L-protein precursor . The derived amino acid sequence indicates that the protein precursor consists of 501 amino acid residues, including a presequence peptide of 31 amino acid residues . The N-terminal sequence of the first 18 amino acid residues of the purified L-protein confirms the identity of the cDNA . Alignment of the deduced amino acid sequence of L-protein with human, porcine and yeast dihydrolipoamide dehydrogenase sequences reveals high similarity (70% in each case), indicating that this enzyme is highly conserved . Most of the residues located in or near the active sites remain unchanged . The results described in the present paper strongly suggest that, in higher plants, a unique dihydrolipoamide dehydrogenase is a component of different mitochondrial enzyme complexes . Confidence in this conclusion comes from the following considerations . First, after fractionation of a matrix extract of pea-leaf mitochondria by gel-permeation chromatography followed by gel electrophoresis and Western-blot analysis, it was shown that polyclonal antibodies raised against the L-protein of the glycine-cleavage system recognized proteins with an Mr of about 60000 in different elution peaks where dihydrolipoamide dehydrogenase activity has been detected . Second, Northern-blot analysis of RNA from different tissues such as leaf, stem, root and seed, using L-protein cDNA as a probe, indicates that the mRNA of the dihydrolipoamide dehydrogenase accumulates to high levels in all tissues . In contrast, the H-protein (a specific protein component of the glycine-cleavage system) is known to be expressed primarily in leaves . Third, Southern-blot analysis indicated that the gene coding for L-protein in pea is most likely to be present in a single copy/haploid genome. Arch Biochem Biophys, 1992 Mar, 293(2), 382 - 90 Relationship between the catalytic center and the primary degradation site of triosephosphate isomerase: effects of active site modification and deamidation; Sun AQ et al.; Covalent modification of the active site Glu165 of triosephosphate isomerase (TPI) (EC 5.3.1.1) with the substrate analogue 3-chloroacetol phosphate (CAP) induces conformational changes similar to those observed during catalysis . We have introduced CAP into the active sites of TPI from yeast, chicken, pig, and rabbit, and assessed the effect of this modification on the structural integrity of the protein . CAP binding accelerated the specific deamidation of Asn71 in mammalian TPI . Transverse urea gradient gel electrophoretic analysis showed that the CAP-TPI dimer dissociates more readily than the native dimer . Hybrids composed of one CAP-modified subunit and one native subunit exhibited intermediate stability . The deamidated enzyme was more susceptible to proteases and denaturing conditions . Subtilisin cleaved the rabbit enzyme primarily at the Thr139-Glu140 bond . The resulting peptides remained noncovalently attached, and the enzyme retained catalytic activity . The data provide further evidence of the interactions between the catalytic center and the subunit interface and that the specific deamidation destabilizes the enzyme initiating its degradation . The enhancement of deamidation upon binding of substrate and catalysis suggest that molecular wear and tear may be involved in regulating proteolytic turnover of the enzyme. Andrologia, 1992 Mar-Apr, 24(2), 87 - 93 Immunosuppression by human seminal plasma fractionated by DEAE Sephadex A-50 ion exchange chromatography; Haq A et al.; We fractionated the whole human seminal plasma on DEAE Sephadex A-50 ion exchange columns . Complete separation was achieved in seven peaks using different salt concentrations in phosphate buffer pH 6 . The seminal plasma proteins were separated by sodium dodecyl-sulphate polyacrylamide gel electrophoresis . Human seminal plasma (SP) and its fractions were used in mixed lymphocyte reaction in vitro . Fractions 3, 4, and 7 were found to suppress the proliferation of human peripheral blood mononuclear cells to phytohemagglutinin and pokweed mitogen at a concentration of 10 micrograms ml-1 while stimulatory effect was observed at lower concentrations (1 microgram and 2.5 micrograms ml-1) . Whole human SP and other fractions failed to suppress the proliferation of lymphocytes in vitro . Furthermore, the effect of human SP and its fractions was also investigated on phagocytic function of polymorphonuclear leukocytes (PMNs) using luminol dependent chemiluminescence assay stimulated with phorbol myristate acetate and opsonized yeast . Fractionated SP was found to have a suppressive effect on the luminol-dependent chemiluminescence of PMNs in the whole blood. J Am Acad Dermatol, 1992 Mar, 26(3 Pt 2), 452 - 7 A double-blind, placebo-controlled, multicenter trial of lithium succinate ointment in the treatment of seborrheic dermatitis . Efalith Multicenter Trial Group. {Antifungal varnish in the treatment of denture stomatitis} Carlino P, Lang R, Budtz-Jorgensen E. Division de Gerodontologie et prothese adjointe, Universite de Geneve, SuisseThe efficacy of a single-dose application of miconazole varnish in the treatment of denture stomatitis was compared with miconazole gel applied three times/day for 15 days . Among 288 wearers of complete dentures 50 patients severely affected by denture stomatitis and heavily colonized by Candida, were selected . The patients were assigned randomly to two groups: a miconazole varnish group and a gel group . All patients were examined 7 times: before starting the treatment (day 0); during treatment (day 3, 7 and 14); after treatment (day 21, 28 and 35) . At each examination a photograph of the palatal mucosa was obtained and quantitative cultures of Candida from the lesions and the fitting denture surface were performed . At day 14 the inflammation was reduced to the same degree in the two groups of patients . A comparison of the antifungal effect, i.e . reduction of the yeast score, showed no significant difference between the two groups . Maximum reduction of the number of yeasts was obtained at day 14 . From that moment, recolonization started; thus, about 60% of the treated patients whether it was with the gel or the varnish was positive of Candida by day 35 . In conclusion, the present study showed no difference in the clinical and antifungal effect of a single-dose application of miconazole varnish compared with miconazole gel . The varnish seemed to be better with respect to the posology as the total dose of miconazole is minimal and only one application is necessary . Treatment with the miconazole varnish seems particularly indicated in debilitated and non-cooperative patients suffering from Candida-associated denture stomatitis. Biochem Cell Biol, 1992 Mar-Apr, 70(3-4), 207 - 14 Mitochondrial activity and heat-shock response during morphogenesis in the pathogenic fungus Histoplasma capsulatum; Patriarca EJ et al.; Changes in temperature and a variety of other stimuli coordinately induce transcription of a specific set of heat-shock genes in all organisms . In the human fungal pathogen Histoplasma capsulatum, a temperature shift from 25 to 37 degrees C acts not only as a signal that causes transcription of heat-shock genes, but also triggers a morphological mycelium- to yeast-phase transition . The temperature-induced morphological transition may be viewed as a heat-shock response followed by cellular adaptation to a higher temperature . We have found that by inducing thermotolerance, i.e., an initial incubation at 34 degrees C, the thermosensitive attenuated Downs strain of H . capsulatum can be made to resemble those of the more temperature-tolerant G222B strain with respect to mitochondrial ATPase activity and electron transport efficiency at elevated temperatures . Furthermore, if the heat-shock response is first elicited by preincubation at milder temperatures or stress, transcription of heat-shock mRNA in mycelial cells of Downs strain that shifted to 37 degrees C proceeds at rates comparable to those of the virulent strains. Genes Dev, 1992 Mar, 6(3), 345 - 55 Expression of an activated Notch-related int-3 transgene interferes with cell differentiation and induces neoplastic transformation in mammary and salivary glands; Jhappan C et al.; Expression of the int-3 locus is activated in mouse mammary tumors as a consequence of insertional mutagenesis by the mouse mammary tumor virus (MMTV) . Integration of the MMTV provirus into the int-3 locus promotes the transcription and translation of flanking cellular int-3 sequences sharing significant homology with the intracellular domain of the neurogenic Notch gene of Drosophila, and with the yeast cell cycle regulatory genes cdc10 and SWI6 . To determine the in vivo consequences of activated int-3 expression, transgenic mice were generated harboring a genomic tumor DNA fragment consisting of the MMTV LTR and the flanking cellular int-3 sequences . All six int-3 founder transgenic mice and the progeny of one established line exhibited similar dramatic phenotypic abnormalities in tissues in which the transgene was expressed . Focal and often multiple poorly differentiated mammary and salivary adenocarcinomas appeared in the majority of transgenic mice between 2 and 7 months of age . Significantly, mammary glands were arrested in development and were lactation deficient in all female int-3 mice . The salivary glands, glands of the nasal mucosa and maxillary sinus, the extraorbital lacrimal glands, and the Harderian glands of juvenile and adult transgenic mice all contained proliferating immature ductule cells and were incompletely differentiated . In addition, all male int-3 transgenic mice were sterile, apparently the result of severe hyperplasia of the epididymis . These findings demonstrate in vivo that expression of the activated Notch-related int-3 gene causes deregulation of normal developmental controls and hyperproliferation of glandular epithelia. Mol Phylogenet Evol, 1992 Mar, 1(1), 59 - 71 Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence; Berbee ML et al.; Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release . Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms . The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes . The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently . Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge . In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates . A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test . Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge . Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA . The transition-to-transversion ratio was 1.9 . Of all 12 possible substitutions, 29% were from U to C . At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4) . In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5). Eur J Biochem, 1992 Mar 1, 204(2), 841 - 6 Bilayer-penetrating properties enable apocytochrome c to follow a special import pathway into mitochondria; Jordi W et al.; In this study, we have investigated the protein/lipid interactions of two mitochondrial precursor proteins, apocytochrome c and pCOX IV-DHFR, which exhibit mitochondrial import pathways with different characteristics . In-vitro-synthesized apocytochrome c was found to bind efficiently and specifically to liposomes composed of negatively charged phospholipids and showed a (at least partial) translocation across a lipid bilayer, as reported previously for the chemically prepared precursor protein {Rietveld, A . & de Kruijff, B . (1984) J . Biol . Chem . 259, 6704-6707; Dumont, M . E . & Richards, F . M . (1984) J . Biol . Chem . 259, 4147-4156} . Negatively charged liposomes were shown to efficiently compete with mitochondria for import of in-vitro-synthesized apocytochrome c into the organelle, suggesting an important role for negatively charged phospholipids in the initial binding of apocytochrome c to mitochondria . In contrast, the purified and in-vitro-synthesized precursor fusion protein pCOX IV-DHFR, consisting of the presequence of yeast cytochrome oxidase subunit IV fused to mouse dihydrofolate reductase was unable to translocate across a pure lipid bilayer . The data indicate that the ability of apocytochrome c to spontaneously translocate across the bilayer is not shared by all mitochondrial precursor proteins . The implications of the special protein/lipid interaction of apocytochrome c for import into mitochondria will be discussed. Nucleic Acids Res, 1992 Feb 25, 20(4), 735 - 45 Molecular cloning of the cDNA for the catalytic subunit of human DNA polymerase delta; Yang CL et al.; The cDNA of human DNA polymerase delta was cloned . The cDNA had a length of 3.5 kb and encoded a protein of 1107 amino acid residues with a calculated molecular mass of 124 kDa . Northern blot analysis showed that the cDNA hybridized to a mRNA of 3.4 kb . Monoclonal and polyclonal antibodies to the C-terminal 20 residues specifically immunoblotted the human pol delta catalytic polypeptide . A multiple sequence alignment was constructed . This showed that human pol delta is closely related to yeast pol delta and the herpes virus DNA polymerases . The levels of pol delta message were found to be induced concomitantly with DNA pol delta activity and DNA synthesis in serum restimulated proliferating IMR90 cultured cells . The human pol delta gene was localized to chromosome 19 by Southern blotting of EcoRI digested DNA from a panel of rodent/human cell hybrids. J Biol Chem, 1992 Feb 25, 267(6), 3589 - 96 The effects of dNTP pool imbalances on frameshift fidelity during DNA replication; Bebenek K et al.; The use of unequal concentrations of the four deoxynucleoside triphosphates (dNTPs) in DNA polymerization reactions alters base substitution error rates in a predictable way . Less is known about the effects of substrate imbalances on base addition and deletion error rates . Thus, we examined pool bias effects on frameshift fidelity during DNA synthesis catalyzed by replicative DNA polymerases . Imbalanced pools altered the frameshift fidelity of the human immunodeficiency virus type-1 reverse transcriptase . Both mutagenic and antimutagenic effects were observed for minus-one, plus-one, and minus-two nucleotide errors, in a highly sequence-specific manner . Most of this specificity can be rationalized by either of two models . One involves frameshifts initiated by pool bias-induced nucleotide misinsertion, and the other involves pool bias-initiated template-primer slippage . Several examples of complex mutations were also recovered more than once in small mutant collections . These contained closely spaced single-base substitution and minus-one base frameshift changes . The two changes occurred at a frequency much higher than predicted if they were generated independently . This suggests that when the polymerase makes one mistake, the probability that it will make a second mistake within the next few incorporations increases significantly . Perturbation of dNTP pools also affected the frameshift fidelity of the replicative yeast DNA polymerase alpha . In reactions containing a low concentration of one dNTP, the error rate increased for one-nucleotide deletions at homopolymeric template nucleotides complementary to the dNTP whose concentration was low . We extended this approach to determine the frameshift fidelity of simian virus 40 origin-dependent semiconservative replication of double-stranded DNA in extracts of human cells . In reactions performed with an equal concentration of all four dNTPs, replication was highly accurate for minus-one-nucleotide errors . However, when the concentration of one dNTP was decreased, the replication error rate increased at complementary, homopolymeric template positions . This response provides an approach for describing frameshift accuracy during replication of the leading and lagging strands. J Biol Chem, 1992 Feb 25, 267(6), 3577 - 80 Lysine 480 is not an essential residue for ATP binding or hydrolysis by Na,K-ATPase; Wang K et al.; Lysine 480 has been suggested to be essential for ATP binding and hydrolysis by Na,K-ATPase because it is labeled by reagents that are thought to react with the ATPase from within the ATP binding site . In order to test this hypothesis, Lys-480 was changed to Ala, Arg, or Glu by site-directed mutagenesis, and the resultant Na,K-ATPase molecules were expressed in yeast cells . The ATPase activity of each of the mutants was similar to the activity of the wild type enzyme indicating that Lys-480 is not essential for ATP hydrolysis . The binding of {3H}ouabain in both ATP-dependent and inorganic phosphate-dependent reactions was used to determine the apparent affinity of each mutant for ATP or Pi . The K0.5(ATP) for ouabain binding to phosphoenzyme formed from ATP was 1-3 microM for Lys-480, Arg-480, and Ala-480, whereas for Glu-480 the K0.5(ATP) was 18 microM . The K0.5(Pi) for ouabain binding to phosphoenzyme formed from inorganic phosphate was 16-28 microM for Lys-480, Arg-480, and Ala-480, but was 74 microM for Glu-480 . The Kd for ouabain binding was similar for both the wild type and mutant Na,K-ATPase molecules (3-6 nM) . These data indicate that the substitution of an acidic amino acid for lysine at position 480 appears to reduce the affinity of the Na,K-ATPase for both ATP and phosphate . It is concluded that Lys-480 is not essential for ATP binding or hydrolysis or for phosphate binding by Na,K-ATPase but is likely to be located within the ATP binding site of the Na,K-ATPase. Nature, 1992 Feb 20, 355(6362), 733 - 5 Dynamin is a GT